Cystic fibrosis lung disease might be corrected by delivering a normal copy of the CFTR cDNA to sufficient epithelia to restore electrolyte and liquid transport. Towards this goal, our Project has developed lentiviral vectors based on feline immunodeficiency virus (FIV) because of their ability to integrate and persist. We also identified a number of virus envelopes (pseudotypes) that confer apical entry on polarized epithelia. The testing of the utility of gene transfer for CF has been hindered by lack of an animal model that recapitulates the human phenotype. Recently, this PPG developed a pig CF model. Animals heterozygous for a CFTR null or AF508 knock in allele were generated. Remarkably, live born CFTR-/- and CFTR[-deltaF508] piglets share many features of human babies with CF including pancreatic insufficiency, meconium ileus, liver involvement, and respiratory abnormalities. We will use this animal model to evaluate the efficacy of lentiviral gene transfer in modifying CF pulmonary disease. There are 3 aims. Aim 1. Which lentiviral pseudotype achieves the best apical targeting and persistent gene transfer? We propose a thorough comparison of several candidate envelopes in pig and human cells to select an optimal vector pseudotype. We hypothesize that one or more pseudotyped lentivirus will target surface airway epithelia that express CFTR and possess progenitor capacity. Aim 2. Can we increase the gene transfer efficiency of the GP64 envelope by directed evolution on airway epithelia? We identified the baculovirus GP64 envelope as having very favorable properties for in vivo airway transduction. We hypothesize that we can further improve lentiviral gene transfer efficiency and specificity by directed evolution of GP64. We will use a reverse genetics system to engineer a replication competent GP64 pseudotyped VSV and perform serial passage on primary pig and human airway epithelia using wild type GP64 to evolve an ainway adapted envelope. In a complementary approach, the GP64 receptor binding domain will be randomly modified by PCR mutagenesis, and this library of novel lentivirus mutants screened on ainA/ay epithelia. These same directed evolution approaches can be applied to other candidates that emerge from Aim 1. Aim 3. Will lentiviral gene delivery correct the CFTR defect and prevent or slow lung disease progression? We will perform gene transfer to the pig ainA/ays using our optimized vectors to identify the pseudotypes with efficient gene transfer and persistence. We hypothesize that FIV gene transfer of CFTR to the ainA/ays of CFTR[Delta F508/Delta F 508] pigs will prevent or significantly slow lung disease progression. Outcome measures will include in vivo bioelectrics, ligh resolution CT scans, screening for infection and inflammation by bronchoscopy, and other measures of lung health.